Purification and characterization of Pseudomonas aeruginosa alkaline phosphatase

Abstract

Alkaline phosphatase and a subunit form of the enzyme have been isolated from Pseudomonas aeruginosa. The enzyme is pure as judged by molecular-sieve chromatography, sodium dodecyl gel electrophoresis, and ultracentrifugation. The enzyme possesses the following properties: (a) existence of three forms: monomer mol. wt. 39 000, dimer mol. wt. 68 000, and tetramer mol. wt. 139 000; (b) pH optimum 10.5; (c) Michaelis constant Km = 6.6 × 10−5 M p-nitrophenyl phosphate; and (d) energy of activation 5647 cal/mol. Amino acid analysis indicates a protein that is hydrophobic. Its physical behavior in solution supports this conclusion. These results explain the observed association of alkaline phosphatase and lipopolysaccharide and substantiate the current theory that the alkaline phosphatase of P. aeruginosa is bound to the outer cell wall in vivo.